孕期DEHP暴露对F1代雄性小鼠睾丸早期发育的影响及机制研究

发布时间：2019-06-05 08:25

【摘要】：邻苯二甲酸二(2-乙基-己基)酯(DEHP)作为环境中丰富存在的内分泌干扰物,易于迁入环境,不易被降解,已受到人们的普遍重视。现有实验证明DEHP会造成神经、甲状腺、肾脏、心脏、生殖发育等多方面毒性,与雌性相比雄性生殖系统对DEHP更为敏感。目前为止,已证明DEHP暴露可以造成雄性个体附睾、睾丸、前列腺的湿重显著降低,肛殖距显著减小,并能引发隐睾等多种生殖损伤。其具体损伤机制包括影响雄鼠睾丸DNA甲基化状态、诱导生精细胞发生凋亡、抑制生精细胞增殖、影响雄激素的合成、诱导生精细胞产生DNA损伤等。本实验在ICR小鼠怀孕的第9.5天到第15.5天通过灌胃进行不同浓度的DEHP暴露(75mg/kg/d,225mg/kg/d,675mg/kg/d),分别在F1代雄性小鼠胚胎期第18.5天以及出生后14、28、35天取材,应用HE染色和透射电镜技术,在组织学水平和超微结构水平研究孕期DEHP暴露对F1代雄性胎鼠和青春期小鼠造成的睾丸病理损伤;并进一步应用Western blot、TUNEL检测、免疫组织化学检测等实验方法,从内质网稳态失衡与内质网应激介导的凋亡、细胞增殖和睾酮合成及代谢等方面探究DEHP造成雄性生殖损伤的具体作用机制。结果发现孕期DEHP暴露后造成:1、胚胎期生精小管数目显著减少,出生后生精小管管间和生精细胞间间隙增大、空泡化、断层、染色异常和脱落的细胞数目增加、生精细胞排列紊乱。2、睾丸生精细胞内质网明显肿胀;内质网应激相关蛋白GRP78相对表达量在出生后14d和28d显著下调,35d显著上调。3、内质网应激介导细胞凋亡的标志分子Cleaved Caspase 12的蛋白相对表达量,在出生后28d和35d显著上调;出生后35d睾丸细胞凋亡数目明显增加;睾丸中PCNA表达阳性的生精小管比例显著下调。4、间质细胞内脂滴数明显减少;部分线粒体形态异常;STARD10的蛋白相对表达量,在胚胎18.5d 675mg/kg/d剂量组显著下调;雄激素结合蛋白ABP1的表达量明显下降。综上所述,孕期DEHP暴露对发育早期的F1代雄性小鼠睾丸会造成比较明显的生殖损伤,可能是通过以下途径造成的:(1)改变内质网的形态结构,扰乱生精细胞内质网稳态,引发内质网应激,并通过内质网应激介导细胞凋亡;(2)抑制生精细胞增殖;(3)影响间质细胞内睾酮合成,降低雄激素在体内的有效浓度等。本研究初步探究了孕期DEHP暴露对F1代雄性小鼠睾丸早期发育的损伤及损伤机制,并首次以小鼠为动物模型将内质网稳态失衡、内质网应激介导的生精细胞凋亡作为DEHP诱导雄性生殖损伤的具体机制之一,为进一步了解人类孕期DEHP暴露与男性后代生殖系统损伤及生殖障碍的关系提供理论基础。
[Abstract]:Bis (2-ethyl-hexyl) phthalate (DEHP), as a rich endocrine disruptor in the environment, is easy to move into the environment and is not easy to be degraded. Existing experiments have shown that DEHP can cause nerve, thyroid, kidney, heart, reproductive development and other toxicity, and the male reproductive system is more sensitive to DEHP than female. So far, it has been proved that DEHP exposure can significantly reduce the wet weight of epididymis, testes and prostate, reduce the anal distance, and cause a variety of reproductive injury such as cryptorchidymis. The specific injury mechanism includes affecting the DNA methylation status of male rat testes, inducing apoptosis of spermatogenic cells, inhibiting the proliferation of spermatogenic cells, affecting the synthesis of androgen, inducing DNA damage of spermatogenic cells, and so on. Different concentrations of DEHP (75mg kg / d, 225mg kg / d, 675mg kg / d) were exposed to different concentrations of DEHP (75mg kg / d, 225mg kg / d, 675mg kg / kg / The samples of F1 generation male mice were collected on the 18.5 day of embryo stage and 14, 28 and 35 days after birth, respectively. HE staining and transmission electron microscope were used. The pathological damage of testes induced by DEHP exposure during pregnancy to F1 generation male fetal mice and adolescent mice was studied at the level of histology and ultrastructure. Furthermore, the steady state imbalance of endoplasmic reticulum and the apoptosis mediated by endoplasmic reticulum stress were further studied by Western blot,TUNEL detection and immunohistochemical detection, and other experimental methods were used to detect the steady state of endoplasmic reticulum and apoptosis mediated by endoplasmic reticulum stress. Cell proliferation, testosterone synthesis and metabolism were used to explore the specific mechanism of male reproductive damage induced by DEHP. The results showed that after DEHP exposure during pregnancy, the number of spermatogenic tubules decreased significantly, the space between seminal tubules and spermatogenic cells increased, vacuolation, tomography, abnormal staining and the number of exfoliated cells increased. The arrangement of spermatogenic cells was disordered. 2, the endoplasmic reticulum of spermatogenic cells in testes was obviously swollen. The relative expression of endoplasmic reticulum stress-related protein GRP78 was significantly down-regulated on the 14th and 28th day after birth, and up-regulated on the 35th day after birth. 3. The relative expression of Cleaved Caspase-12, a marker of apoptosis mediated by endoplasmic reticulum stress, was significantly up-regulated on the 28th and 35th day after birth. On the 35th day after birth, the number of apoptosis of testicular cells was significantly increased, the proportion of spermatogenic tubules with positive expression of PCNA in testes was significantly down-regulated. 4, the number of lipid droplets in Leydig cells was significantly decreased, and some of the mitochondria were abnormal. The relative expression of STARD10 protein was significantly down-regulated and the expression of androgen-binding protein ABP1 was significantly decreased in 18.5 days of embryo 675mg/kg/d dose group. To sum up, DEHP exposure during pregnancy can cause obvious reproductive damage to the testes of F1 male mice in the early stage of development, which may be caused by the following ways: (1) changing the morphology and structure of endoplasmic reticulum and disturbing the homeostasis of endoplasmic reticulum in spermatogenic cells. Endoplasmic reticulum stress was induced and apoptosis was mediated by endoplasmic reticulum stress. (2) inhibit the proliferation of spermatogenic cells, (3) affect testosterone synthesis in Leydig cells, reduce the effective concentration of androgen in vivo and so on. In this study, the damage and mechanism of early testicular development in F1 male mice induced by DEHP exposure during pregnancy were investigated, and the steady state imbalance of endoplasmic reticulum in mice was used as an animal model for the first time. Endoplasmic reticulum stress-mediated spermatogenic cell apoptosis is one of the specific mechanisms of male reproductive injury induced by DEHP, which provides a theoretical basis for further understanding the relationship between DEHP exposure during pregnancy and reproductive system injury and reproductive disorder in male offspring.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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